The Ministry of Health and Welfare Notification No. 1997-22 Guidelines for researches involving recombinant DNA molecules in accordance with the provisions of Article 15 of the Biotechnology Promotion Law and Article 15 of the Enforcement Ordinance of the same law shall be established as follows. April 22, 1997 Minister of Health and Welfare Guidelines for researches involving recombinant DNA molecules Chapter 1. General Provisions Article 1 (Purpose) The purpose of these guidelines is to prevent occurrence of any biological hazard resulting from propagation and dissemination of biotechnological mutants, and to promote biotechnological research by specifying experimental practices to ensure the safety of researches involving recombinant DNA molecules and its experiments according to Article 15 of the Biotechnology Promotion Law and Article 15 of the Enforcement Ordinance of the same law. Article 2 (Definitions) The following terms, which are used throughout the Guidelines, are defined as follows: 1. "Recombinant DNA molecules" means DNA molecules that are constructed in vitro by joining heterologous DNA molecules to DNA molecules(vector) that can replicate in certain livning cells using enzymes, etc.. 2. "Researches involving recombinant DNA molecules" means experiments that replicate heterologous DNA by transferring recombinant DNA molecules into cells and experiments that use cells containing recombinant DNA molecules. 3. "Experiments in accordance with the researches involving recombinant DNA molecules" means the experiments that replicate DNA by transferring heterologous DNA into cells of animals or plants without using a vector for the purpose of differentiation in researches involving recombinant DNA molecules. 4. "Organisms containing recombinant DNA molecules (referred to as recombinants hereinafter)" means cells or organisms that have parts of their genes exchanged or have newly transferred genes as a result of researches involving recombinant DNA molecules and experiments in accordance with researches involving recombinant DNA molecules. 5. "A host" means the cell that recombinant DNA molecules are transferred into in researches involving recombinant DNA molecules. 6. "A vector" means the DNA that transports heterologous DNA into the host in researches involving recombinant DNA molecules. 7. "A host-vector system" means the joining of a host and a vector.
Transcript
The Ministry of Health and Welfare Notification No. 1997-22
Guidelines for researches involving recombinant DNA molecules in accordance with the provisions of Article 15 of the Biotechnology Promotion Law and Article 15 of the Enforcement Ordinance of the same law shall be established as follows.
April 22, 1997Minister of Health and Welfare
Guidelines for researches involving recombinant DNA molecules
Chapter 1. General Provisions
Article 1 (Purpose) The purpose of these guidelines is to prevent occurrence of any biological hazard resulting from propagation and dissemination of biotechnological mutants, and to promote biotechnological research by specifying experimental practices to ensure the safety of researches involving recombinant DNA molecules and its experiments according to Article 15 of the Biotechnology Promotion Law and Article 15 of the Enforcement Ordinance of the same law.
Article 2 (Definitions) The following terms, which are used throughout the Guidelines, are defined as follows: 1. "Recombinant DNA molecules" means DNA molecules that are constructed in vitro by joining heterologous DNA molecules to DNA molecules(vector) that can replicate in certain livning cells using enzymes, etc.. 2. "Researches involving recombinant DNA molecules" means experiments that replicate heterologous DNA by transferring recombinant DNA molecules into cells and experiments that use cells containing recombinant DNA molecules. 3. "Experiments in accordance with the researches involving recombinant DNA molecules" means the experiments that replicate DNA by transferring heterologous DNA into cells of animals or plants without using a vector for the purpose of differentiation in researches involving recombinant DNA molecules. 4. "Organisms containing recombinant DNA molecules (referred to as recombinants hereinafter)" means cells or organisms that have parts of their genes exchanged or have newly transferred genes as a result of researches involving recombinant DNA molecules and experiments in accordance with researches involving recombinant DNA molecules. 5. "A host" means the cell that recombinant DNA molecules are transferred into in researches involving recombinant DNA molecules. 6. "A vector" means the DNA that transports heterologous DNA into the host in researches involving recombinant DNA molecules. 7. "A host-vector system" means the joining of a host and a vector. 8. "DNA donors" means organisms that supply DNA needed to be inserted into a vector. It includes organisms that supply RNA when DNA reverse-transcribed from RNA template is inserted into vectors. 9. "Large scale cultivation experiment" means experiment involving recombinant DNA molecules using fermenter of larger than 50 liters. 10. "Recombinant animal" means the animal prepared by the following parameters: (It includes a fertilized egg, an embryo, a fetus, an adult, and cultured cells with parts of preceding subjects and cells cultured for the purpose of differentiation. Same shall apply hereinafter.) a) Experiments that use animals as hosts b) Transforming experiments in which recombinants are transferred into animals. c) Experiments that use animals prepared by the parameters as in (a) and (b) 11. "Recombinant plant" means the plant prepared by the following parameters: (It includes a pollen, a spore, a seed, and cultured cells with differentiated parts of preceding subjects, and cells cultured for the purpose of differentiation. Same shall apply hereinafter.) a) Experiments that use plants as a host b) Transforming experiments that recombinants are trasnferred into plants c) Experiments that use plants prepared by the parameters as in (a) and (b)
12. "A laboratory" means the room in which researches involving recombinant DNA molecules are conducted. 13. "Research area" means the area such as a laboratory, a hallway, etc. that is isolated from the other area in order to control the access. 14. "Special research district" means the confidential district with safety control system installed within. 15. "Isolated outdoor research district" means the specific district such as an isolated farm, and it is the place in which experiments such as evaluation of safety, etc. are executed. 16. "Non-isolated district" means the specific district such as a greenhouse or a screened room, etc. that is different from isolated district such as research area, special research district, etc., and it is the place in which experiments such as evaluation of safety, etc. are executed before performing experiments in isolated outdoor research district. 17. "A safety cabinet" means the equipment that has the structure and size at least equal to the equipment as in Appendix 1 that is designed to prevent release of contaiminated aerosols, etc. during the experiment. 18. "Laboratory staff" means persons engaging in researches involving recombinant DNA molecules. 19. "Principal Investigator" means the person among laboratory staff who is responsible for each research procedure.
Article 3 (Classification of Experiments) Researches involving recombinant DNA molecules and researches in accordance with researches involving recombinant DNA molecules (referred to as "experiments", hereinafter) are classified as follows depending on the category of organisms used as a host-vector system or DNA donors. 1. Experiments involving culture cells, etc. a) Experiments using microorganisms and cultured cells, etc. as hosts These experiments use microorganisms(including Rickettsia and Chlamydia and excluding virus, etc. Same shall apply hereinafter) and cultured cells(confined to those without the purpose of differentiation) as a host. The DNA molecules originated from virus, etc. in the host cells control the expressions as a promoter and terminator, etc., without producing infectious virus particles. b) Experiments using animals or plants as hosts These experiments use animals, plants or cultured cells(confined to those used for the purpose of differentiation) as hosts. The DNA molecules originated from virus, etc. in the host cells control the expressions as a promoter and terminator, etc., without producing infectious virus particles. 2. Experiments involving virus, etc. These experiments use virus of eukaryotes(excluding fungi), viroid and protozoa of vertebrates. Except for those cases the DNA molecules originated from virus, etc. in the host cells control the expressions as a promoter and terminator, etc., without producing infectious virus particles.
Article 4 (Procedures to Ensure the Safety of Experiments) Üç Experiments are classified as follows according to the procedures necessary for ensuring the safety. 1. Non-standard Experiments It is the experiments that does not fall under the containment conditions determined in the Guidelines. It is to be approved by the Head of Institution about the research plan, and it is to be executed under the guidance of the head of the authority involved. 2. Standard experiments a) Experiments which requires approval by institution It is the experiments that falls under the containment conditions determined in the Guidelines, and it shall be approved by the Head of Institution. b) Experiments which requires report to institution It is the experiments that falls under the containment conditions deternined in the Guidelines, and its plan shall be reported to the Head of Institution before execution. Üè Non-standard experiments and experiments which requires approval by institution among standard experiments shall be investigated by the Institutional Biosafety Committee established in institution concerned in accordance with the provision of Article 20.
Article 5 (Establishment of Experimental Safety) Üç In order to ensure the experimental safety, based on the methods used in general microorganisms laboratory, two kinds of containments such as physical containment and biological containment shall be combined in a proper manner before executing the experiment.
Üè The details of the containment of experiments as in Paragraph 1 shall follow the standard determined in the Guidelines, and each institution shall determine its own regulations to follow.
Chapter 2. Containment
Section 1. Physical containment
Article 6 (Purpose of Physical Containment) The physical containment is to provide means of containment of recombinants within the equipment in order to prevent propagation and dissemination to the laboratory staff or to other objects.
Article 7 (Kinds of Physical Containment) The containment equipment, design of a laboratory and regulations according to the kinds of physical containment are as follows: 1. Physical containment of experiments in the scale smaller than 50 liters a. P1 level 1) Containment equipment and design of a laboratory The laboratory shall be designed to have the following basic equipment of a general microorganism laboratory. Ýw The laboratory shall have the list of the laboratory staff, who are allowed to have periodical access to the laboratory, at the entrance of the laboratory. Ýx The laboratory shall be designed so that it can be easily cleaned and disinfected. The bench tops shall be impervious to water and resistant to strong acids, strong alkalis, organic solvents and disinfectants. Ýy When mechanical ventilation system is installed in a laboratory, a ventilation equipment fitted with fly screens with free airflow shall be installed as well. Especially the case in which airflow of the laboratory is suspended, air supplying and ventilating systems shall be connected to prevent positive pressure. Ýz The laboratory shall contain a sink for hand washing. Ý{ The laboratory shall have a clothing change room. Ý| Mechanical pipetting devices shall be installed. Ý} The disinfectant shall be placed somewhere accessed to treat any contamination during the experiments. Ý~ The facilities to safely store pre-disinfected laboratory apparatus and equipment shall be installed. Ý‘ The facilities to safely store any wastes produced during the experiments shall be installed. Ý’ Wastes that are to be treated in an autoclave or an incinerator shall be placed in a durable leak-proof container which is closed before removing from the laboratory. 2) Regulatory rules Ýw All laboratory staff shall be well-informed about the experimental procedures and safety of the experiments. Ýx The bench tops shall be decontaminated after the experiments. Any contamination during the experiments shall be immediately decontaminated. Ýy All contaminants originated from the experimental organisms shall be sterilized before disposal, and other contaminated apparatus shall be re-used after cleansing or decontaminated or sterilized before disposal. Ýz Laboratory windows and doors shall be kept closed when experiments are in progress. Ý{ Mechanical pipetting devices shall be installed. Ý| Eating, drinking, smoking, applying cosmetics, and storing food are not permitted in the work area. Ý} Persons shall wash their hands after handling materials involving recombinants and when exiting the laboratory. Ý~ All procedures shall be performed carefully to minimize the creation of aerosols. Ý‘ Used experimental apparatus and equipment shall be disinfected, and safely stored until disinfection. Used pipets shall be immersed completely in disinfectant. Ý’ The contaminated materials shall be safely stored before decontamination in an autoclave, and an incinerator. Ý“ Contaminated materials that are to be discharged at a site away from the laboratory shall be placed in a durable leak-proof container which is closed before being removed form the laboratory. Ý” An insect and rodent control program shall be in effect.
Ý• Needles shall not be used when other methods are available. Ý– Using laboratory clothing shall follow the instruction of the Principal Investigator. Ý— Other rules determined by the Principal Investigator shall be obeyed. b. P2 Level 1) Containment equipment Ýw In using a blender, lyophilizer, sonicator, or centrifuge with high potential for creating aerosols, for processing recombinants, the contaminated aerosols shall not escape. Ýx Safety cabinet shall be used, and the integrity of the cabinet shall be investigated if necessary. 2) Design of the laboratory The autoclave shall be installed to treat contaminated materials in the laboratory. 3) Regulatory rules Ýw Laboratory clothing shall be worn in the laboratory, and changed when leaving the laboratory. Ýx Persons without the knowledge of the experiments in progress shall not be allowed to enter the laboratory without the permission of the Principal Investigator. Ýy "P2 level experiments in progress" sign shall be posted on the access doors to the laboratory. The sign shall also be posted on the freezer and refrigerator storing the recombinants. Ýz The HEPA filter of the safety cabinet shall be decontaminated by formaldehyde fumigation before exchanging or during investigation. Ý{ When executing P1 level experiments in the same laboratory, clear district shall be decided before the execution. Ý| P1 level regulatory rules and rules determined by the Principal Investigator shall be obeyed. c. P3 Level 1) Containment equipment Ýw When handling recombinants, use of equipment producing aerosols shall be carried out in the safety cabinet, except when using aerosol leak-proof equipment. Ýx When installing safety cabinet, periodical investigation, periodical exchange of HEPA filter, formaldehyde fumigation shall be executed without moving the safety cabinet. 2) Design of laboratory Ýw Research area shall be territorialized.And the anteroom shall be so designed that not both of the doors may be opened simultaneously. Clothing room shall be included. Ýx The autoclave shall be installed to treat contaminant and wastes. Ýy The surfaces of floor, wall and ceiling shall be in a structure, made of materials which may be easily cleaned and fumagated. Ýz A foot, elbow, or automatically operated hand washing sink shall be provided in the laboratory and near the door of each laboratory room. Ý{ The laboratory windows shall be closed and sealed. Ý| The doors in the research area shall be automatically operated. Ý} The experimental vacuum inhalation equipment, and the suction hole shall be installed with filter or trap that uses disinfectant solution. Ý~ The research area shall be equipped with air ventilation equipment. 3) Regulatory rules Ýw Laboratory clothing with long sleeves, pull-on without buttons shall be worn in the laboratory only. It shall be disinfected before washing. Ýx Access to the research area shall be through anteroom. Ýy When the experiments is in progress, a "P3 level experiments in progress" sign shall be posted on the entrance, and the freezer, refrigerator, etc. storing recombinants shall have the same sign posted. Ýz Laboratory gloves shall be worn in the laboratory, and the used gloves shall be decontaminated after completing the work. Ý{ The experiments that requires contaminant level lower than P2 shall not be concomitantly performed in this laboratory. Ý| Regulatory rules of P2 level and the rules determined by the Principal Investigator shall be obeyed. D. P4 level 1) Containment equipment Ýw Safety cabinet such as Class ÛB to handle recombinants shall be installed. However, ventilation shall be done by the life-maintaining system within the special research area. When the
experiments is performed wearing laboratory clothing that can maintain positive pressure, the safety cabinet of Class Û@ or Class ÛA may be used instead. Ýx Periodical investigation, exchange of HEPA filters, formaldehyde fumigation, etc. shall easily be done without moving safety cabinet. 2) Design of the laboratory Ýw Research area shall be located in P4 level-only building or in area strictly separated from other areas within the building, and persons other than laboratory staff shall not be authorized to enter. Ýx The anteroom shall be so designed that not both of the doors may be opened simultaneously, and a clothing room and a shower room shall be installed. Ýy In case of bringing in reagents or materials to the research area wihout passing through the clothing room, the reagents or materials shall be brought in through double-doored anteroom. Ýz The floor, wall and ceiling shall have the structure that allows fumigation and be insect and rodent proof. All penetrations shall be sealed to maintain constant pressure during fumigation. Ý{ The reserach area shall be self-closing and locking. Ý| A foot, elbow, or automatically operated hand washing sink shall be provided in the laboraory and near the principal exit of each laboratory room and reasearch area in the facility. Ý} Central vacuum system for reserch area shall be installed, and HEPA filter shall be installed in each area and near the investigation cork and HEPA filter shall be allowed to be decontaminated and exchanged without dissembling. Ý~ An apparatus for back flow proof shall be installed in the ducts supplying water, gas, etc.. Ý‘ In order to decontaminate materials disposed from the research area, an autoclave which is so designed that not both of the doors may be opened simutaneously (referred to as "autoclave" hereinafter) shall be used. Ý’ When materials disposed from the reserach area cannot be heat-decontaminated, sedimentation tank filled with disinfectant solution(referred to as "sedimentation tank" hereafter) or fumigation chamber which is so designed that not both of the doors may be opened simultaneously (referred to as "fumigation chamber", hereinafter) shall be installed. Ý“ Fast ventilation equipment shall be installed for the research area only, and in case air is brought in from outside, pressure difference shall be maintained to allow the outside air to flow into the areas of higher hazard level and to prevent back flow. Alarming equipment shall be installed to detect any disorder and malfunction of the equipment. Ý” Each laboratory shall have its air recirculated through HEPA filter. Ý• Ventilation from the research area shall go through HEPA filter and then to the ventilator of the nearby building. The HEPA filter shall be decontaminated and exchanged without dissembling. Ý– The exhaust air from the Class ÛB safety cabinet shall be disposed outside, and the air treated from Class Û@ and Class ÛA safety cabinet may be disposed within the laboratory. However, when disposing through ventilator within the research area, air balance within the safety cabinet and ventilation system shall be maintained. Ý— The special experimental district shall satisfy the following parameters. a) Life-maintaining equipment shall have alarm and emergency air tank installed. b) Airlock of the laboratory door shall be installed. c) Chemical shower room to decontaminate any contaminant on clothing shall be installed. d) Ventilation of the district shall be filtered twice through HEPA filters. e) Two types of ventilation shall be used for safety. f) Emergency electric-power source, light and communication equipment shall be installed. g) Negative pressure shall always be maintained in the research area other than the assigned district. h) Autoclave shall be installed to decontaminate any wastes discharged from the assigned district. 3) Regulatory rules. Ýw Biological materials to be removed from the class ÛB safety cabinets or to be brought in to the class ÛB safety cabinets from outside in a viable or intact state shall be transferred to a non-breakable, sealed container which is removed from the facility or brought in to the facility through a sedimentation tank or fumigation chamber. Ýx Materials, except for biological materials that are to remain in a viable or intact state, shall be removed from or brought in to the class ÛB safety cabinets after they have been autoclaved. Equipement or material which might be damaged by high pressure and steam, shall pass through sedimentation tank or fumigation chamber.
Ýy Personnel shall enter and exit the facility through the anteroom, and shower each time they enter and exit the facility. Ýz Complete laboratory clothing, including undergarments, pants and shirts or jump suits, shoes, head covers, and gloves, shall be worn in the research area. When exiting the research area and before proceeding into the shower room, personnel shall remove their laboratory clothing and store it in a hamper. Ý{ A biological hazard warning sign incorporating the universal biosafety symbol shall be posted on all access doors to the laboratory work area, and freezers or refrigerators in which recombinants are stored. Ý| Liquid wastes from safety cabinets, and laboratory sinks shall be decontaminated by heat treatment, and liquid effluent from shower rooms and toilets shall be sterilized or decontaminated with chemical disinfectants. Ý} Experiments of contaminant level lower than P3 level shall not be concomitantly performed in the laboratory concerned. Ý~ Regulatory rules of P3 level and other rules determined by the Principal Investigator shall be obeyed. 2. Physical containment of large scale cultivation experiments. a. LS-C level 1) Contaminant equipment and its design Ýw A closed system used for the propagation and growth of cultures(referred to as an incubator hereinafter), other equipment and apparatus shall be kept in good state. Ýx Containment equipment shall be designed to minimize the release of recombinants through gas effluent from the incubator. 2) Regulatory rules Ýw Eating, drinking, applying cosmetics and storing foods are not permitted in the district to perform large scale cultivation experiments(referred to as "large scale cultivation experiments district" hereafter). Ýx All wastes originated from viable organisms related to large scale cultivation experiments shall be inactivated before disposal by a validated inactivation procedure. A validated inactivation procedure shall be demonstrated to be effective using the organism that will serve as the host for a large scale cultivation experiments. Ýy When inoculating recombinants in an incubator or when collecting recombinants from the incubator as a reagent, special care shall be taken not to contaminate outer surface of the incubator. Ýz When moving recombinants to other equipment or apparatus from an incubator, contamination by exposure to the recombinants shall be minimized. Ý{ The large scale cultivation experiments district shall be kept clean, and an insect and rodent control program shall be in use. Ý| The incubator with large scale cultivation experiments in progress shall have "Large scale cultivation experiments of LS-C level in progress" sign posted. Ý} When using large scale cultivation experiments laboratory clothing, instructions given by the Principal Investigator shall be followed. Ý~ Other regulatory rules determined by the Principal Investigator shall be obeyed. b. LS-1 level 1) Containment equipment and its design Ýw The laboratory shall be designed so that overt exposures to recombinants can be prevented and the incubator in which internal sterilization can be done in closed state shall be installed. The incubator shall have its containment degree investigated every year and right after the installation. Ýx In using a blender, lyophilizer, sonicator, or centrifuge with high potential for creating aerosols, for processing recombinants, a class ÛA safety cabinet or containment equipement at least equal to the class ÛA safety cabinet (referred to as "class ÛA safety cabinet, etc." hereinafter) Ýy Safety cabinet shall have its condition investigated every year and right after the installation. Ýz Exhaust gases removed from an incubator of recombinants shall be treated by filters which have efficiencies equivalent to high efficiency particulate air/HEPA filters (referred to as "HEPA filters, etc." hereinafter). HEPA filters, etc. shall have their conditions investigated every year and right after the installation. Ý{ When reconstructing or exchanging the sealed parts, sealing and function of the appropriate equipment shall be investigated. 2) Regulatory rules. Ýw Large scale cultivation experiments district shall clearly be territorialized.
Ýx An incubator or other contaminated equipment and apparatus, and all wastes originated from viable organisms related to large scale cultivation experiments shall be inactivated before disposal by a validated inactivation procedure. A validated inactivaton procedure shall be demonstrated to be effective using the organism that will serve as the host for a large scale cultivation experiments. Ýy Mechanical pipetting is recommended. Ýz After handling recombinants and when exiting large scale cultivation experiments area, hands shall be washed. Ý{ All procedures shall be performed carefully to minimize the creation of aerosols. Ý| When inoculating recombinants in an incubator or when collecting recombinants from the incubator as a reagent, special care shall be taken not to contaminate outer surface of the incubator. In case of contamination, immediate disinfection shall be done. Ý} When moving recombinants from one incubator to other incubtor and when collecting recombinants from the incubator as a reagent, the recombinants shall be kept in a sturdy, leak-proof container. The outer surface of the container shall not be contaminated, and in case of contamination, immediate disinfection shall be done. Ý~ When removing culture liquid containing recombinants from the incubator (excluding safety cabinet) or disposing it to outside, a validated sterilization proceure shall be taken. A validated sterilization procedure shall be demonstrated to be effective by using the organism that will serve as the host for a large scale cultivation experiments. Ý‘ Contaminated materials that are to be decontaminated at a site away from the large scale cultivation experiments area shall be placed in a durable leak-proof container which is to be closed before being removed from the area. Ý’ The large scale cultivation experiments area shall be insect and rodent free. Ý“ "LS-1 level large scale cultivation experiments in progress" sign shall be posted on the access doors to the laboratory in which large scale cultivation experiments is in progress. The sign shall also be posted on the freezer and refrigerator storing recombinants. Ý” Use of large scale cultivation experiments laboratory clothing shall be according to the instruction of the Principal Investigator. Ý• Containment conditions of an incubator shall be examined at least once a day when the large scale cultivation experiments is in progress. Ý– Decontamination filters of the safety cabinet and other equipment shall be decontaminated before its exchange or during periodical investigation. Ý— Separate district is required when concomitantly executing other experiments of P1 level containment. (16) Other rules determined by the Principal Investigator shall be obeyed. C. LS-2 level 1) Containment Equipment and its Design Ýw The laboratory shall be designed so that overt exposures to recombinants can be prevented, and the incubator shall be equipped so that inner sterilization can be performed in closed state. Ducts and other parts connected to the incubator shall be designed so that release of recombinants may be precluded. Ýx After installing the incubator, and every time large scale research is held, the integrity of containment shall be evaluated. Ýy In using a blender, lyophilizer, sonicator, or centrifuge which has high potential for creating aerosols, for processing recombinants, a class ÛA safety cabinet or containment equipment at least equal to a class ÛA safety cabinet (referred to as "class ÛA safety cabinet, etc." hereinafter) is required. However, using devices which are designed lest aerosols be released to outside may be permitted. Ýz Exhaust airs of an incubator containing recombinants shall be discharged through filters or other equipment which can remove microorganisms. Capacity of Filters and other equipment used for removing microorganisms shall be evaluated immediately after installation, and annually. Ý{ Class ÛA safety cabinets, etc. shall be installed so that periodical examinations, exchanges of filters, formaldehyde fumigation can be performed without moving class ÛA safety cabinets, etc. Ý| Containment equipment for an incubator and other tools directly connected to it, and class ÛA safety cabinets, etc. are equipped with sensors for monitoring containment degree. Ý} All equipment and apparatus shall be kept in records on examination, operation, and tagged serial distinguishing number. Ý~ The laboratory shall be placed in a building equipped with autoclaves which disinfect contaminants or wastes.
Ý‘ In case parts related to containment are needed to be repaired or exchanged, containment degree and the function of apparatus or devices concerned shall be evaluated. 2) Regulatory rules Ýw Laboratory coats, gowns, smocks, or uniforms shall be worn in the laboratory. Before entering non-laboratory areas, protective clothing shall be removed. Ýx An individual ignorant of the substance of the large scale research shall not be allowed to enter or leave the laboratory without permission of the Principal Investigator. Ýy When large scale research is in progress, a "Large scale research of LS-2 level in progress" sign shall be posted on access doors to the laboratory, and on refrigerators and freezers where recombinants are stored, a sign indicating the presence of recombinants shall be posted. Ýz When large scale research is in progress, the current containment situation of the incubator and tools directly connected to it, and class ÛA safety cabinets, etc. shall be continuously monitored with a sensor. Ý{ When filters of class ÛA safety cabinets or other devices are exchanged or periodically examined, or there is any change in large scale research, the equipment shall be sealed, and then after formaldehyde fumigation of 10 g/ÜC, the equipment shall be left for one hour as it is, or shall be decontaminated by other handling methods with functions at least equal to the preceding method. Ý| If experiments which require P1 or P2 level of containment, or other large scale research of LS-1 level are conducted simulatneously, the area shall be clearly territorialized and experiments shall be conducted prudentially. Ý} Laboratory staff shall observe regulatory rules of LS-1 level or matters designated by the Principal Investigator.
Section 2. Biological Containment
Article 8 (Purpose of Biological Containment) The purpose of biological containment is, in case of an experiments involving virus, etc., to preclude the possibility of transfer, or dissemination of recombinants into the environment by using a host-vector system in which the host not surviving in conditions other than in a specific culture condition and the vector having no infectivity to other non-laboratory hosts than to the specific host are combinated together, and to maintain the biological safety of recombinant DNA high and to secure the safety of an experiments by using a host-vector system which is recognized as having very high biological safety, and in case of an experiments involving culture cells wihch has higher level of safety than an experiments involving virus, etc., to secure the safety of the experiments by reviewing the biological properties of DNA donors in the process of recombinant formation and the biological properties of the composition of host-vector system.
Article 9 (Levels of Biological Containment) Üç Levels of biological containment are classified in accordance with kinds of experiments as following. 1. Levels of biological containment in experiments involving virus, etc., are classified into B1 and B2 in accordance with the biological safety of host-vector system(hereinafter, host-vector systems on level equal to or lower than B1 or B2 are referred to as "certified host-vector systems"). The description of each level is as follows, and host-vector systems under each level are designated by the head of appropriate authority. a. B1 level The level of biological containment of host-vector systems which have recognized potential to preclude the possibility of transfer, and dissemination of recombinants into the environment by combination of a host with low viability in nature and a vector hard to propagate to other cells due to its high dependence on the host, or host-vector systems which are recognized as having high level of biological safety to human beings based on their genetic, and physiological properties, or their ecology in the natural world b. B2 level The level of biological containment of host-vector systems which satisfy the conditions of B1 level, and also have recognized potential to preclude the possibility of transfer and dissemination of recombinants into the environment by using the combination of a host with exceptionally low viability in nature and a vector with exceptionally high dependence on the host. 2 .In case of experiments involving culture cells, the levels of biological containment shall be designated after overall judgement of each following item concerning the host-vector system and the biological properties of DNA donor.
a. Pathogenicity b. Productivity of toxins c. Parasitic/fixing property d. Carcinogenicity e. Drug resistance f. Effect on the metabolic system g. Effect on the ecosystem h. Dependence on the host i. Transferring property Üè In case of experiments using host-vector systems in which animals, plants or culture cells(limited to those not intended to be used for differentiation) of exceptionally high level of safety are used as hosts and there appear no infective virus particles, the host-vector systems can be treated as belonging to B2 level, under the guidance of the head of appropriate authority.
Chapter 3. Containment Conditions
Article 10 (Containment conditions) Üç Basic containment conditions concerning examination on safety of recombinants are as follows. 1. The containment conditions for recombinants are designed according to the combination of examination on safety by biological properties of host-vector systems and DNA donors. 2. Among host-vector systems and DNA donors in use, what needs the highest level of containment is used in designating the containment conditions of recombinants according to examination on safety in principle. However, in case of transferring vectors, close attention to safety shall be paid in addition. 3. Experiments involving certified host-vector systems are considered as having high safety level in comparison with other experiments. 4. In large scale experiments, among DNA derived from cells, etc., cloned DNA, and synthetic DNA, DNA with clear functions, size and structure and with useful functions like production of useful proteins, etc. shall be used. 5. In case that experiments which need P1 or P2 level of physical containment are performed in scale larger than 50 liters, LS-1 or LS-2 level of physical containment shall apply for each case. 6. In case of experiments using recombinants whose exceptionally high biological safety has been verified among those large scale research which fall under Paragraph 4 or Paragraph 5, special physical containment methods other than LS-C level of physical containment or physical containment method according to the ordinance of Article 7 may apply under the guidance of the head of authority concerned. 7. For the safety of large scale research, complete experimental facilities with various kinds of containment equipments including fermentation equipment on a large scale or more than equally equipped facilities are used, but in case of large scale culture experiments using recombinants of verified high safety level like LS-C level, facilites with well-equipped large scale culture equipment, etc. and more than equally equipped facilities may be used. 8. In raising recombinant animals or growing recombinant plants, matters designated in Table 1 shall be observed. Üè Irrespective of the provisions of Paragraph Üç, in case of using DNA with clear functions, size and structure among DNA derived from cells, cloned DNA, and synthetic DNA, the levels of containment may be lowered after reviewing following items by the Institutional Biosafety Committee. 1. Pathogenicity 2. Productivity of toxins 3. Carcinogenicity 4. Transferring property
Table 1 : Details needed to be observed in raising animals or growing plants{{{{The raising and managementof recombinant animals}}{{The cultivation and management of recombinant plants}}{{Üç Facilities preventing animals from escaping shall be installed in the access doors, ventilators, drainage, windows, etc. The access doors shall be closed in cases other than entrance or exit.
Windows shall not be opened (in case of animal experiments, only laboratory staff can open), and there shall be equipment which prevents opening from outside.Üè The cage shall be strong enough not to be open or broken easily by the animals' strength and swinging.Üé Animals shall be distinguished from each other as much as possible. In case that it is hard to distinguish animals (insects, fish) from each other, animals shall be raised in a cage respectively, and the cages shall be clearly distinguised from those of animals without recombinants concerned.Üê Straw, excretion and water shall be disinfected or incinerated, if necessary.Üë An insect and rodent control program shall be in use.Üì In case of carrying animal outside of the laboratory, the animal shall be put into a solid container clearly described of which the structure impedes escape of the animal even though it is broken.Üí A sign of "Recombinant animal experiment in progress" shall be posted on the access doors to the laboratory.Üî An individual who enters the animal room shall pay attention not to contaminate the room with insects, etc. carried through clothes or containers in entering or exiting.
}}{{Üç If necessary, plants shall be separately cultivated using plant cultivating equipment of closed type (hereinafter, referred to as plant cultivating equipment) like bottles, plastic boxes, etc., and in other cases, plants shall be cultivated in an open area (greenhouse). In case of trees which require many years for blooming, however, cultivation in an open area outdoors is possible until efflorescence. However, in case of cultivation of these trees in an open area, facilities preventing individuals ignorant of the purpose of the cultivation and wild beasts from approaching the experiment area shall be installed.Üè Ventilation of the laboratory or plant cultivating equipment shall be performed after wrapping with paper to prevent pollens, etc. (refer to spores or seeds. The same shall apply hereinafter.) from scattering.Üé Insects which may transmit pollens, etc. shall be ridded.Üê The recombinant plants shall not be exposed through draining water, and drainage shall be disinfected by autoclave, if necessary.Üë In handling wastes or soil related to the plants, the wastes or soil shall be inactivated by using methods with verified efficiency.Üì In case that plants are carried out of the laboratory, necessary precautions against plants slipping out shall be taken.Üí A "Recombinant plant experiment in progress" sign shall be posted on the access doors of the laboratory.}}}}
Üé Classification table of safety level of culture cells as provided for under Item 2, Paragraph 1 of Article 9, is the same as Appendix 2, Appendix 3, and Appendix 4, and Detailed standards for classification of experiments in accordance with the steps related with Article 4 is the same as Appendix 5. And Containment standards table in accordance with the classification of experiments related with Article 3 is the same as Appendix 6, Appendix 7, Appendix 8, and Appendix 9.
Chapter 4. Handling of Recombinants
Article 11 (Storage of recombinants) When storing recombinants, follwing items shall be observed. 1. Samples and/or wastes containing recombinants shall be clearly indicated as "Recombinants", and stored safely in a laboratory, experiments area, or large scale research area satisfying physical containment conditions designated for the experiments using the recombinants. 2. On refrigerators or freezers in which samples and wastes containing recombinants are stored, "Storing recombinants" shall be marked. 3. The Principal Investigator shall draw up and keep papers of samples and wastes containing recombinants. In case of samples and/or wastes (limited to those containing recombinants. Same shall apply hereinafter.) which need physical containment levels equal to or lower than P2 level, experiments data may be allowed as a substitute.
Article 12 (Transport of recombinants) When transporting recombinants, following items shall be observed. 1. In case of carrying samples and wastes which need physical containment equal to or lower than P2 level out of a laboratory, they shall be carried out of the laboratory after putting them into a sturdy leak-proof container, and then, sealing the container. 2. In case of carrying samples and wastes which need physical containment equal to or higher than P3 level out of a laboratory or research area, matters as provided for under Article 13 shall be observed, and the container shall be made not to leak even though it is damaged, and on the container or the surface of packing material, "Handle with care" shall be written clearly in red. 3. The Principal Investigator shall draw up and keep documents of names of transported recombinants, quantities, name of organization transporting recombinants (name of institution and name of Principal Investigator). However, recording of recombinants which need physical containment equal to or lower than P2 level may be allowed as a substitute. 4. In carrying samples and wastes which need physical containment of LS-C level out of large scale research areas, such handling as in case of physical containment equal to or lower than P2 level shall apply. 5. In case of samples and wastes which need physical containment of LS-1 and/or LS-2, such handling as in case of physical containment equal to or higher than P3 level shall apply.
Article 13 (Assignment of recombinants) Üç Recombinants may be assigned to other institution. Üè An institution which has received recombinants in accordance with the provision of Paragraph 1 shall observe steps designated in Article 4.
Article 14 (Handling of recombinants after experiments) Üç After experiments are finished, recombinants shall be inactivated before disposal. In case that experiments other than an experiments related need to be conducted with a recombinant concerned, the recombinant can be preserved by reporting papers on the completed experiments and the Principal Investigator to the head of institution. Üè In case that other experiments need to be performed with a recombinant made from a standard experiments, the experiments shall be guided by head of authority involved.
Chapter 5. Training and Health Care
Article 15 (Training) In order to secure safety of an experiments, the Principal Investigator shall train laboratory staff up to a necessary level in the following items. 1. Skills in treating microorganisms safely according to their biohazard potential. 2. Knowledge and skills of physical containment 3. Knowledge and skills of biological containment 4. Information of biohazard potential of an experiments to be performed 5. Knowledge of emergency plans (In case culture fluid containing recombinants is leaking in large scale research, appropriate measures like sterilization by chemical processing, etc. shall be taken.)
Article 16 (Health Care) In order to secure safety of an experiments, the head of institution shall provide laboratory staff with following items related to health care. 1. If deemed necessary, laboratory staff shall undergo periodical medical surveillance. 2. In case that infection is suspected in a laboratory or large scale research area, medical examination shall be taken immediately, and appropriate advance-measures or ex post facto measures shall be taken. 3. Results of medical examinations shall be written down and kept at least for two years, or in case of laboratory staff equal to or higher than P3 level, at least for five years. However, laboratory staff engaging in only experiments equal to or lower than P1 level, shall be exempt. 4. If laboratory staff fall into one of the following items, or have been reported in accordance with the provision of Paragraph 2 of Article 17, prompt examinations and necessary measures shall be taken. a. When a laboratory staff have drunk or breathed in recombinants by mistake b. When the skin is contaminated with recombinants
c. When a person was in a laboratory, research area, or large scale research area contaminated remarkably by recombinants
Chapter 6. Measures for the Purpose of Securing Safety of Experiments
Article 17 (Laboratory Staff) Üç Laboratory staff shall perfectly understand matters on securing safety in planning or performing an experiments, and shall be trained in general experimental methods of microorganisms, special handling skills of the experiments, and related experiments. Üè In case that a laboratory worker feels strange about his body, or has been suffering from frequent or chronic diseases, he shall report to the head of institution, and if another person has perceived this fact, he shall report in the same way. Üé A laboratory worker shall confirm that a host-vector system which is used in an experiments satisfies biological and physical containment conditions during the experiments as well as before starting the experiments.
Article 18 (The Principal Investigator) The Principal Investigator shall perfectly understand matters determined by these guidelines, and shall perform each of following duties as an expert who has a good stock of knowledge and technology of preventing disasters caused by viable organisms and related knowledge and technology including them. 1. Concerning initiation of an experiments plan and performing an experiments, the Principal Investigator shall observe matters determined by the guidelines completely, and perform appropriate management and supervision. 2. The Principal Investigator shall plan and perfom training to laboratory staff in accordance with Paragraph 15. 3. Concerning standard experiments and experiments which requires approval by institution (hereinafter, change in experiments plan is included), the head of Institution shall submit an experiments plan to the head of authority concerned. 4. Concerning experiments which requires to institution, an experiments plan shall be reported to the head of institution in advance. 5. The Principal Investigator shall observe matters concerning storage and transport of recombinants determined in Article 11 and Article 12. 6. Other matters necessary for securing safety of an experiments shall be performed. Üè The Principal Investigator shall keep in touch with the Institutional Biosafety Committee sufficiently during an experiments and report to the Institutional Biosafety Committee about necessary matters.
Article 19 (the Head of Institution) The Head of Institution is a person responsible for securing safety of experiments, and shall, 1. establish and operate the Institutional Biosafety Committee, and assign the members 2. perform matters concerning training laboratory staff and health care by taking the Institutional Biosafety Committee's advice 3. in case of large scale research, preserve each data as follows after experiments is finished for the period determined by the head of authority concerned, and submit data to the head of autority concerned when asked. However, in case of experiments of LS-C level, recording of experiments may be replacement a. Data which is evidence confirming that large scale experiments is suitable for the guidelines b. Investigation record of the Institutional Biosafety Committee c. Data on matters concerning securing safety among experiments facilities, experiments methods, and experiments results 4. in case of standard experiments, perform experiments after experiments is evaluated by the head of authority concerned through inspections by the Institutional Biosafety Committee, and submit papers on results(interim report) of safety inspection 5. in case of experiments requiring approval by institution, determine suitability through the the Institutional Biosafety Committee's inspections 6. in case of experiments that requires reporting to institution, accept experiments plans 7. in case of storage of recombinants, accept papers on completed experiments, and report on storage place and the Principal Investigator 8. take necessary measures when ethical problem occurs
9. perfom necessary matters on securing safety of other experiments
Article 20 (The Institutional Biosafety Committee) Üç The Institutional Biosafety Committee. in accordance with the provision of Item 1, Article 19 must be comprised of members so selected that they have experience and expertise. and in order to secure safety of experiments performed in institution, shall inspect, examine following items, and consult the head of institution. 1. Suitability of experiments plan to the quidelines 2. Training in experiments and health care 3. Necessary measures and/or reform measures in case of accidents 4. Matters needed in preventing ethical problems 5, Other necessary matters concerning securing safety of experiments Üè The Institutional Biosafety Committee can ask report from the Principal Investigator if deemed necessary.
Article 21 (Governmental guidance) The head of authority related shall secure safety of recombinant DNA research activities, and for the purpose of effective operation of the guidelines, can perform matters on following items. 1. Organization and operation of a conference for the purpose of consulting with the authority 2. Examination and authoritive analysis of standard experiments by consultation with conference in accordance with Item 1 above. 3. Guidance for the purpose of securing safety of experiments 4. Prevention of ethical problems
Article 22 (The Recombinant DNA Advisory Committee) Üç In order to provide advice and suggestions requested by the Minister of Health and Welfare, the Recombinant DNA Advisory Committee may be installed under the Ministry of Health and Welfare. Üè The Recombinant DNA Advisory Committee is composed of experts with knowledge and expertise in the field of biotechnology who are recommended by authorities concerned. Üé The Recombinant DNA Advisory Committee shall examine matters of following items and advise the Minister of Health and Welfare. 1. Collective settlement of executive matters on duties of the head of authority concerned in accordance with the provision of Article 21. 2. Matters on examination of national or overseas information in accordance with provisions of Article 25 and Article 26. 3. Matters necessary for preventing ethical problems 4. Other matters concerning securing safety of experiments Üê Irrespective of the provision of Paragraph 1, the Recombinant DNA Advisory Committee can advise the head of authority concerned if deemed necessary.
Article 23 (Prevention of Ethical Problems) The head of authority concerned and the head of institution shall devise proper measures for preventing occurrence of ethical problems by banning experiments which are apt to cause harm on human dignity like transfer of recombinant DNA molecules into the genome of one or more human subjects.
Chapter 7. Examination Procedures
Article 24 (Application of examination, etc.) Üç The Head of Institution who wants to obtain examination of standard experiments shall submit an application for the examination of the experiments to the head of authority concerned in accordance with Attachment Û@. Üè The head of authority concerned who has received an application in accordance with the provision of Paragraph 1 shall review data on examination application of experiments of Appendix 10, and after determining the suitability, announce the head of Institution. Üé The head of Institution who is announced in accordance with the provision of Paragraph 2 shall submit a result report on experiments of safety examination to the head of authority concerned one month after the end of the experiments, attaching result or interim result data on safety examination experiments of Appendix 11, following Attachment ÛA, and if deemed necessary, can instruct to submit an intermediate report for the purpose of securing safety of the experiments.
Article 25 (Exchange of international information) Üç The Ministry of Health and Welfare is the major authority for exchanging internatinal information necessary for securing safety of genetic recombination. Üè The Minister of Health and Welfare who has collected international information in accordance with the provision of Paragraph 1 shall take proper measures including announcing to the head of authority involved, and the head of authority concerned shall take proper measures to apply this effectively.
Article 26 (Handling of domestic information) Üç For the purpose of effective handling of safety information of domestic experiments, the head of authority concerned shall examine the safety information obtained by performing the Guidelines, and then announce it to the Minister of Health and Welfare. Üè The Minister of Health and Welfare shall take proper measures including announcement to the head of authority concerned, and the head of authority concerned shall take measures to apply this effectively.
Article 27 (Supplementary Rule) In order to secure safety of genetic recombination experiments, the head of authority concerned may designate detailed guide including methods for securing safety of each classification and the examination methods in the scope of the Guidelines.
Addendum
1. (Enforcement Date) The Guidelines shall come into force three months after notice.
Appendix 1. Safety Cabinets and Standards of HEPA Filters (Related to Item 17 of Article 2)Class Û@{{{{Usage}}{{For treating microorganisms or pathogens of low or moderate biohazard potential in work area not requiring clean air.}}{{Design, standard }}{{There are a full-width open front and an exhaust duct. The inward flow of air from the full-width open front precludes release of contaminated air, and the exhaust air from this cabinet is filtered through a high efficacy air/HEPA filter and released outside of the cabinet. The face velocity of the inward flow of air through the full-width open front is 0.40 m/sec or greater.}}}}
Class ÛA{{{{Usage}}{{For treating microorganisms or pathogens of low or moderate biohazard potential in work area requiring filtered air. It is used for general biological purpose(type A), and for treating aerosols or materials which are hard to be seived by a HEPA filter.}}{{Design}}{{There are a full-width open front and an exhaust duct. The inward flow of air from the full-width open front precludes release of contaminated air, and HEPA-filtered air is provided. The cabinet exhaust air is filtered through a HEPA filter and discharged to the outside environment. A cabinet of which the positive pressure contaminant plenum is in contact with exterior walls among type A cabinets ise not recommended. Of type B, exhaust air shall be discharged to outdoors via ducts connected to the cabinet.}}{{Standard}}{{Containment degree
when applying pressure to the inside of the cabinet by 50 mmHg, internal pressure decline shall be less than 10 percent. When spraying soapy water or formy agent for detecting leakage to all parts of the cabinet and ducts, there shall be no detection of leakage (For a cabinet whose positive pressure plenum is in contact with outer walls, halogen leakage rate shall be lower than 5 ÙO 10-7Ü6/sec)
Safety testingIn spraying 5Ù=10 ÙO 108 cfu baccillus spore, the number of colonies collected by dust collecting equipment shall be less than 10. Five to fifteen minutes after starting the experiments, number of collected colonies in slit sampler shall be less than 5 each time of experiments. This test shall be successful in three successive experiments.
Sample protection testingIn spraying 5Ù=10 ÙO 106 cfu baccillus spore, colony number collected in agar plate (filling Petri dish of diameter of 10 centimeters as much as possible. Same shall apply hereinafter) shall be less than five each time of experiments. Three successive testings shall be successful.}}}}{{{{}}{{Testing of prevention of mutual contamination between samplesIn spraying 5 Ù= 10 ÙO 104 baccillus spore, colony number collected in an agar plate of which the center is away from the side by more than 35 mm shall be less than 2 in total. Three successive testings in both sides shall be successful.
Airflow velocityAirflow velocities in each part of a grid less than 15 cm shall be within the scope of ÙN20% of average velocity. In case of a cabinet where differential airflow velocity is possible, airflow velocity shall be calculated in parts designated by the manufacturer.
Inflow velocityAverage inflow velocity from the open-width front shall be higher than 0.40 m/s.(in case of type B, higher than 0.50 m/s)
BlowerA blower shall be designed so that decline of blow quantity is less than 25 percent even without controlling rotation when the pressure loss of the filter is more than 20 percent.
Direction of airflowDirection of airflow is decided by examination of inflow state to the smoke generating duct with eyes. Smoke shall flow smoothly downward when injecting air from two spots of each side of work zone. One spot is located at the front and back airflow dust of vertical laminar airflow of the work zone, and is located higher than the lower end of the front panel by at least 100ÙN10mm. And the other spot is located higher than the lower end of the front panel by at least 150ÙN20mm and is interior to the front panel by 20ÙN30 mm. There shall be no part where smoke does not flow, or smoke flows upward. And smoke shall not escape from the cabinet. When injecting smoke from 30Ù=40mm outside of the open-width front to the open-width front, smoke shall neither leak nor flow into the work zone.
Increase in temperatureDifference between room temperature and the temperature inside of the cabinet shall be less that 8ÙY after continuous operation.
Noise levelNoise level shall be less than 67dBA.
IlluminanceMean illuminance shall be 80Ù=1200 lux.
VibrationThe scope of vibration of three orthogonal direction of the work zone shall be less than 5Ü= RMS.
LiquidA support for the liquid (collector) shall be designed so as to ensure easy cleaning, and have capacity of at least four liters.}}}}{{{{Consideration oncleaning andsterilization}}{{The surface which is apt to contaminated by liquid, its particle, drops, etc. shall be designed to be cleaned without tools. The work table and work zone shall be round edged. The structure shall be constructed so as to fumigate with formaldehyde without moving the body. The open-width front, exhaust duct shall be able to be sealed with metal plates, plastic sheets , and adhesive tapes. The sole of the cabinet shall be designed to be higher than the floor by at least 80mm or to adhere closely to the floor or to the prop so that it can easily be cleaned.}}{{Examination}}{{On-the-spot examination of HEPA filter shall be performed once a year and rignt after the installation, since there could be changes such as blocking of HEPA filter which directly influence on performance of HEPA filter.}}}}
Class ÛB{{{{Usage}}{{For treating microorganisms or pathogens of extremely high biohazard potential}}{{Design, standard }}{{It is a closed-front ventilated cabinet of gas tight construction. All supply air and exhaust air are filtered through HEPA filters. Exhaust air is filtered through two HEPA filters or one HEPA filter and incinerator before being discharged to the outside environment. The cabinet is operated under a negative pressure of at least 15 mmHg in comparison to that in the laboratory, and is fitted with arm-length rubber gloves, and autoclaves which are used for sterilizing agents and apparatus with high pressure and temperature, or sinks.}}}}
Right after the installation, the safety cabinet shall be examined on items a) through c), and be examined at least once a year periodically on items a) and b). a) Testing of airflow velocity, airflow power b) Performance testing of HEPA filter c) Containment degree test
HEPA filters related to safety cabinet{{{{Functions, etc.}}{{When examining HEPA filter by loading test aerosol to the dirty part of HEPA filter, the penetration rate of prefixed microsector (the concentration ratio of aerosol in the clean part to that in the dirty part) shall not exceed 0.01%.
In injection testing using relative densimeter or particle counter which sucks 28.3Ü4/min in constant suction condition, the penetrating rate of aerosol of 0.03Ü= or so shall be confirmed not to exceed 0.01% in equipped state.
Installation of manometer to indicate loss of pressure of HEPA filter is required.}}}}
Appendix 2. Classification of microorganisms used in experiments on the basis of hazard (Including rickettsia and chlamydia, and excluding protozoa)
Corynebacterium C. bovis C. diphtheriae C. ovis/pseudotuberculosis C. ulcerans C. renale
Cryptococcus C. neoformansErysipelothrix E. rhusiopathiaeExophiala E. dermatitidisEscherichia E. coli (All enterotoxigenic serotypes)Fusobacterium F. necrophorumFomsecaea F. pedrosoiHaemophilus H. ducreyi
H. influenzaeKlebsiella K. pneumoniaeLegionella All speciesLeptospira L interrogans (all serotypes)Listeria L. monocytogenesMycobacterium M. africanum
Microorganisms not falling into (1) and (2). However, microorganisms whose human pathogenicity has not been proven yet shall be exempt.
Appendix 3. Classification of virus and viroid of eukaryotes(excluding fungi) used as DNA donors in experiments on the basis of hazard (Related to Paragraph 3, Article 10 )
(1) Requiring P3-B1 or P2-B2 containment when used as DNA donors
California encephalitis virus Chikungunya virus Hemorrhagic fever with renal syndrom virus Herpes ateles virus Herpes saimiri virus Hog cholera virus HIV (Human immunodeficiency virus) *1 SIV (Simian immunodeficiency virus) *2 Japanese encephalitis virus La Crosse virus LCM virus Monkeypox virus Murray Valley encephalitis virus O'nyong-nyong virus Powassan virus Rabies (street) virus St. Louis encephalitis virus Tacaribe virus Vesicular stomatitis virus West Nile virus *1 HTLV-III=HIV-1, HTLV-IV=HIV-2, LAV-1=HIV=1, LAV-2=HIV-2 *2 STLV-III is included in SIV.
(2) Requiring P2-B1 or P1-B2 containment when used as DNA donors.
(4) Not requiring containment level when used as DNA donors
Those not falling into (1), (2) and (3)
Appendix 4. Classification of protozoa of vertebrates used as DNA donors in experiments on the basis of hazard (Related to Paragraph 3, Article 10)
(1) Requring P3-B1 or P2-B2 containment when used as DNA donors.
None
(2) Requring P2-B1 or P1-B2 containment when used as DNA donors.
Balantidium B. hominis Entamoeba E. histolytica Giardia G. lambria Hartmanella All species Leishmania L.braziliensis
L. donovani L. mexicana L. peruviana L. iropica Naegleria All species Piroplasma All species Plasmodium P. falciparum P. malariae P. ovale P. vivax
Simian malarial parasites Sarcocystis S. suihominis Toxoplasma T. gondii Trypanosoma T. cruzi T. ganbiense T. rhodesiense
(3) Requiring P1-B1 or P1-B2 containment when used as DNA donors.
Babesia B. bovis Chilomastix C. mesnili Cryptosporidium All species Dientamoeda D. fragilis Enbadomonas E. intestinalis Endolimax E. nana Entamoeba E. coli
E. gingiralis E. hartmanni Enteromonas E. hominis Iodamoeba I. buetschii Isospora I. belli I. hominis Histomonas All species Eimeria All species
Leucocytozoon All species Pneumocystis P. carinii Plasmodium P. berghei Retortamonas R. intestinalis Sarcocystis S. hominis S. lindemanii Torichomonus T. tenax T. hominis T. vaginalis Trypanosoma T. brucei T. rangeli
(4) Not requiring containment level when used as DNA donors.
Those not falling into (1), (2) and (3)
Appendix 5. Detailed classification of experiments on the basis of procedures (Related to Paragraph 3, Article 10){{{{Classification}}{{Non-standard experiments}}{{Standard experiment}}{{Institution-approved}}{{Requiringreport to
institution}}{{Experiments usingmicroorganisms orculture cells as ahost}}{{1. Experiments using microorganisms whose species name and human-pathogenicity have not been identified yet2. Experiments using microorganisms of P3 level as a host or a vector3. Cloning experiments of gene expressing albuminoid toxicity to vertebrate4. Large scale cultivation experiments (except in case of executing in smaller than 50 liters scale, a large scale cultivation experiments executing in larger than 50 liter-scale for the experiments requiring P1 and P2 level of containment, and experiments using recombinants with efficient functions such as useful protein productivity and high biological safety, among DNA derived cells, cloned DNA and chemically synthesized DNA)5. Experiments including dissemination of recombinants to nature}}{{Excludingexperimentrequiringreport toinstitutionandnon-standard experiment
}}{{Experiment using a plant andmicroorganism of P1 levelas a DNAdonor, among the experimentsusing acertified host-vector system
}}{{Experiments usinganimal or plantas a host.}}{{1. Experiments using microorganisms whose species name and human-pathogenicity have not been identified yet2. Experiments using microorganisms of P3 level as a vector3. Experiments producing albuminoid toxicity to the vertebrates4. Experiments that recombinants prepared in non-standard experiments make animal or plant transformed
5. Experiments executing outdoor control of growing and breeding of recombinant animal or plant}}{{Experiment excluding non-standard experiment
}}{{None
}}}}{{{{Classification}}{{Non-standard experiments}}{{Standard experiment}}{{Institution-approved}}{{Requiringreport toinstitution}}{{Experimentsusing virus, etc.}}{{1. Experiments using microorganisms whose species name and human-pathogenicity have not been identified yet2. Experiments involving a host-vector system other than certified host-vector system3. Cloning experiments of gene having albuminoid toxicity on vertebrate4. Experiments inoculating recombinants into animal or plant5. Large scale cultivation experiments (except in case of executing in smaller than 50 liter-scale, a large scale cultivation experiments executing in larger than 50 liter-scale for the experiments requiring P1 and P2 level of containment, and experiments using recombinants with efficient functions such as useful protein productivity and high biological safety, among DNA derived cells, cloned DNA and chemically synthesized DNA).6. Experiments using natural distribution of recombinants}}{{Experiments excluding non-standard experiments
}}{{None
}}}}
Appendix 6. Containment level in experiments using microorganisms and culture cell as a host (In case of executing in smaller than 50 liter-scale) (Related to Paragraph 3, Article 10){{{{ DNA donorHost-vectorsystem}}{{Animal(P1)}}{{Plant(P1)}}{{Appendix2-(1)(P3)}}{{Appendix2-(2)(P2)}}{{Appendix
2-(3)(P1)}}{{EK2 system}}{{P1}}{{Experimentsrequiringreport toinstitution(P1)}}{{P2}}{{P1}}{{Experimentsrequiringreport toinstitution(P1)}}{{EK1 systemSC1 systemBS1 system}}{{P1}}{{Experimentsrequiringreport toinstitution(P1)}}{{P3}}{{ P2}}{{Experimentsrequiringreport toinstitution(P1)}}{{ Culture cell[host] (Only for those not intended for differenti- ation)}}{{Appendix 2-(1)(vector)
Appendix 2-(2)(vector)
Appendix 2-(3)(vector)}}{{Non-standardexperiments
P2
P1
}}{{Non-standardexperiments
P2
P1
}}{{Non-standardexperiments
P3
P3
}}{{Non-standardexperiments
P2
P2
}}{{Non-standardexperiments
P2
P1
}}{{Using Appendix 2-(1) as either host or vector}}{{Non-standardexperiments}}{{Non-standardexperiments}}{{Non-standardexperiments}}{{Non-standardexperiments}}{{Non-standardexperiments}}{{Using Appendix 2-(2) as either host or vector(only for those notusing Appendix 2-(1))}}{{P2}}{{P2}}{{P3}}{{P2}}{{P2}}{{Host-vector systemcomposed ofAppendix 2-(3) only}}{{P1}}{{P1}}{{P3}}{{P2}}{{P1}}}}
- Non-standard experiments for the experiments using microorganisms whose species name and human-pathogenicity have not been identified yet.- Institution-approved experiments for the experiments with only containment level specified. - Appendix 2, Appendix 3 and Appendix 4 will be applied for those having expression control function such as promoter, terminator, etc. of DNA originated from all virus used, without producing infectious virus particles.Ùh 1. EK1 : Host-vector system using genetically and biochemically well-identified, nontoxic E.Coli K 12, with low vitality in natural condition and its derivative as a host, and plamid or bacteriophage
that cannot be transferred to other due to its nonconjugatability as a vector. In this case, the host will have a conjugable plamid or general introductory bacteriophage. 2. SC1: Host-vector system using yeast, S. cerevisiae, laboratory-preserved strain as a host, and plamid or small chromosome as a vector. 3. BS1 : Host-vector system using B. subtilis Marburg 168 derivative, multiple auxotrophic mutants for aminoacid or nucleic acid as a host, and plamid or bacteriophage due to non-conjugatability not transferred to other bacteria as a vector. 4. EK2 : Host-vector system combining a host satisfying EK1 condition as well as having extremely low vitality in conditions other than specific cultivation due to its genetic defects, with a vector of high host-dependency and extremely low trasmittance to other cell.
Appendix 7. Containment level in experiments involving microorganism and culture cell as a host (Large scale cultivation experiments) (Related to Paragraph 3, Article 10){{{{ DNA donorHost-vectorsystem}}{{Animal(P1)}}{{Plant(P1)}}{{Appendix2-(1)(P3)}}{{Appendix2-(2)(P2)}}{{Appendix2-(3)(P1)}}{{EK2 system}}{{LS-1}}{{LS-1}}{{LS-2}}{{LS-1}}{{LS-1}}{{EK1 systemSC1 systemBS1 system}}{{LS-1}}{{LS-1}}{{Non-standardexperiments}}{{LS-2}}{{LS-1}}{{ Culture cell[host] (Only for those not intended for differenti- ation)}}{{Appendix 2-(1)(vector)
}}{{Using Appendix 2-(1) as either host or vector}}{{Non-standardexperiments}}{{Non-standardexperiments}}{{Non-standardexperiments}}{{Non-standardexperiments}}{{Non-standardexperiments}}{{Using Appendix 2-(2) as either host or vector(only for those notusing Appendix 2-(1))}}{{Non-standardexperiments}}{{Non-standardexperiments}}{{Non-standardexperiments}}{{Non-standard
- DNA originated from DNA donor is confined to those having specifically effective function.- Non-standard experiments for the experiments using microorganisms whose species name and human-pathogenicity have not been identified yet.- Institution-approved experiments for those described in containment level only- Appendix 2, Appendix 3 and Appendix 4 will be applied for for those having expression control function such as promoter, terminator, etc. of DNA originated from all virus used, without producing infectious virus particles.Appendix 8. Containment level in experiments using animal or plant as a host (Related to Paragraph 3 Article 10){{{{ DNA donorHost-vectorsystem}}{{Animal(P1)}}{{Plant(P1)}}{{Appendix2-(1)(P3)}}{{Appendix2-(2)(P2)}}{{Appendix2-(3)(P1)}}{{Animal
}}{{Non-standard experiments}}{{Non-standard experiments}}{{Non-standard experiments}}{{Non-standard experiments}}{{Appendix 2-(2)[vector]}}{{P2}}{{P2}}{{P3}}{{P2}}{{P2}}{{Appendix 2-(3)[vector]}}{{P1}}{{P1}}{{P3}}{{P2}}{{P1}}{{Directmethod(Experimentsnot using avector)}}{{P1}}{{P1}}{{P3}}{{P2}}{{P1}}}}
- Non-standard experiments for the experiments using microorganisms whose species name and human-pathogenicity have not been identified yet.- Institution-approved experiments for those described in containment level only- Appendix 2, Appendix 3 and Appendix 4 will be applied for those having expression control function such as promoter, terminator, etc. of DNA originated from all virus used, without producing infectious virus particles.
Appendix 9. Containment level in experiments using virus, etc. (Related to Paragraph 3, Article 10){{{{ Host-vector system DNA donor}}{{B1 host-vectorsystem}}{{B1 host-vectorsystem}}{{Experiments usinghost-vector system otherthan certified host-vectorsystem}}{{Appendix 3-(1) or Appendix 4-(1)(P3-B1)(P2-B2)}}{{P3}}{{P2}}{{Non-standard experiments}}{{Appendix 3-(2) orAppendix 4-(2)(P2-B1)
- Non-standard experiments for those using virus with unidentified species name and human-pathogenecity as DNA donor.- Institution-approved experiments for those described in containment level only.
Appendix 10. Documents required for application evaluating non-standard experiments{{{{Name of project}}{{}}{{Purpose of experiments}}{{}}{{Summary of experiment plan}}{{}}{{Classification of experiments}}{{}}{{Combination of host-vector system and itscharacteristics or its type
}}{{}}{{Biological containment method of host-vector system}}{{}}{{Necessity of host-vector system}}{{}}{{Procedures in preparing host-vector system and origin, characteristics and introduction method of heterologous DNA.}}{{}}{{Characteristics of recombinants}}{{}}{{DNA donors}}{{Kinds of viable organismssupplying DNA donors}}{{}}{{Characteristics in classification}}{{}}{{Pathogenicity, carcinogenicity,and productivity of toxins}}{{}}{{Charateristics of albuminoidtoxicity on vertebrates}}{{}}{{Drug-sensitivity}}{{}}{{Parasitic/Parabiotic}}{{}}{{Distribution in nature}}{{}}{{Origin, and history of itscontact with human}}{{}}{{Kinds of DNA}}{{}}{{Kinds of DNA used}}{{}}{{Attributesof vectorused}}{{Name}}{{}}{{Origin}}{{}}{{Drug resistance}}{{}}{{Transmittance andhost-dependency}}{{}}{{Characteristics}}{{}}}}
{{{{Characteristics ofhost used ororganism speciesthat belongs tothis}}{{Name of host}}{{}}{{Distribution in nature
}}{{}}{{Genetic characteristics}}{{}}{{Gene exchange range and kinds of genes exchanged}}{{}}{{Pathogenicity, carcinogenicity,and productivity of toxins}}{{}}{{Parasitic/Parabiotic}}{{}}{{Origin and historyof its contact with human}}{{}}{{Animal or plants,etc. that are transformed by recombinants}}{{Necessity of experimentinoculating recombinants intoanimal or plant}}{{}}{{Kinds of animal or plants used}}{{}}{{Characteristics of animal orplant used}}{{}}{{Characteristics of recombinantanimal and recombinant plantobtained by inoculatingrecombinants}}{{}}{{Level of physicalcontainment}}{{Lower than 50 liters}}{{P1}}{{P2}}{{P3}}{{P4}}{{Other}}{{Higer than 50 liters}}{{LS-C}}{{LS-1}}{{LS-2}}{{Other}}{{}}{{Opinion on evaluation of safety}}{{}}{{Containment of breeding and growing control ofrecombinant animal or recombinant plant}}{{}}{{Facilities of physical containment }}{{Location}}{{}}{{Structure}}{{}}{{Facility}}{{}}}}
Appendix 11. Document of results or interim results of safety evaluation{{{{Name of project
}}{{}}{{Classification of experiments}}{{Experiments usingmicroorganisms andculture cells as a host
Experiments usinganimal and plant asa host}}{{Experimentsinvolving virus,etc.}}{{DNAdonors}}{{Combination of host-vector system,and its kinds}}{{}}{{DNAdonors}}{{Kinds of DNA donor production}}{{}}{{Kinds of DNA}}{{}}{{Kinds of DNA used}}{{}}{{Animal and plant into whichrecombinants are transferred}}{{}}{{Level of physical containment}}{{ P1 P2 P3 P4 LS-C LS-1 LS-2 Other}}{{Characteristics of recombinants and recombinant plant, etc. obtained by transferring recombinants}}{{}}{{Opinions on evaluation of safety of the experiments}}{{}}{{Post plan of the experiments, Decision on thenecessity to preserve recombinants and/or storingmethod of recombinants, disposal method ofrecombinants.}}{{}}{{PaperPublished}}{{Time of presentation}}{{}}{{Scope of the presentation}}{{}}{{Related journals}}{{}}{{Opinions}}{{}}}}
Attachment Û@{{{{Application of evaluation of non-standard experiments}}{{Transaction period}}{{30 days}}{{Institution(applicant)}}{{Name
}}{{}}{{Address}}{{(Zip code : )}}{{Name of theRepresentative}}{{(Phone No. : )}}{{Name of the project}}{{(New, Continued, Revised)}}{{PrincipalInvestigator}}{{Rank}}{{}}{{Name}}{{(Phone No. : )}}{{Address}}{{}}{{Laboratory}}{{Name}}{{}}{{Address}}{{}}{{Phone No.}}{{}}{{LaboratoryStaff}}{{Name}}{{Position}}{{Experience inhandlingmicroorganisms}}{{Experience in researchesinvolving recombinant DNAmolecules}}{{}}{{}}{{}}{{}}{{InstitutionalBiosafetyCommittee}}{{Head ofthecommittee}}{{Name}}{{(Signature)}}{{Rank}}{{}}{{Reason theexperiment isappropriate}}{{}}{{I heareby apply for the evaluation of non-standard experiments according to Article 24 of the Guidelines for researches involving recombinant DNA molecules.
Date :
Applicant (Signature)
Esq.}}{{Required document : A copy of "Data of application of non-standard experiments evaluation"
}}{{Submission Fee}}{{None}}{{}}}}
Attachment ÛA{{{{Report of results or interim results of safety evaluation}}{{Institution(reporter)}}{{Name}}{{}}{{Address}}{{(Zip code : )}}{{Name of theRepresentative}}{{(Phone No. : )}}{{Name of the project}}{{(New, Continued, Revised)}}{{Experimental Period}}{{ From To}}{{PrincipalInvestigator}}{{Name of theInstitution}}{{}}{{Name}}{{(Phone No. : )}}{{Address}}{{}}{{Laboratory}}{{Name}}{{}}{{Address}}{{}}{{Phone No.}}{{}}{{LaboratoryStaff}}{{Name}}{{Position}}{{Name}}{{Position}}{{}}{{}}{{}}{{}}{{InstitutionalBiosafetyCommittee}}{{Address}}{{(Zip code : )}}{{Head ofthecommittee}}{{Name ofthe Institution}}{{
}}{{Name}}{{(Signature)}}{{I heareby report results or interim results of safety evaluation according to Article 24 of the Guidelines for researches involving recombinant DNA molecules.
Date :
Reporter (Signature)
Esq.}}{{Required document : Document of results or interim results of safety evaluation}}}}
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